Our studies are focused on the expression of growth hormone and prolactin genes in normal and transformed pituitary cells. These experiments are designed to show how post-transcriptional methylation of mRNA molecules affects the processing of nuclear mRNA precursors into mature cytoplasmic mRNAs. Recent progress on this project has occurred in two major areas. First, we have developed a new approach for determining the location of naturally occurring N6-methyladenosine (m6A) residues within specific mRNA sequences. This technique should be broadly applicable because it does not rely on prior in vivo labeling of the mRNA. Using this approach we have determined the location of m6A in bovine prolactin mRNA and found that these modified nucleotides arecontained within a short 3' terminal fragment (129 nucleotides) of this 915 nucleotide-long mRNA. The nonrandom location of m6A residues within prolactin mRNA suggests a specific function which can now be assessed by a combination of techniques involving DNA-mediated gene transfer and site-directed mutation of the bovine prolactin gene. Secondly, by using an inhibitor which blocks mRNAmethylation, we were able to follow the fate of undermethylated mRNA in HeLa cells. Our results indicate that once undermethylated mRNA reaches the cytoplasm, it is degraded at the same rate as fully methylated mRNA. However, the nuclear processing and/or transport of mRNA into the cytoplasm is significantly affected by undermethylation. These nuclear effects can now be studied using individual mRNA sequences, including prolactin and growth hormone mRNA. (G)